Downregulation of endogenous TRAIL and its effect on the human cancer cell line KELLY [Elektronische Ressource] / Sara Brittingham. Betreuer: Irmela Jeremias

Sara Brittingham

Description

Aus der Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen Kinderspital der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. med. Ch. Klein Abteilung Hämatologie/Onkologie Downregulation of endogenous TRAIL and its effect on the human cancer cell line KELLY Dissertation zum Erwerb des Doktorgrades der Medizin an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Sara Brittingham aus Warschau 2011 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: PD Dr. Irmela Jeremias Mitberichterstatter: PD Dr. Andreas Jung Prof. DrFiedrich-Wilhelm Kreth Dekan: Prof. Dr. Dr. h.c. M. Reiser; FACR, FRCR Tag der mündlichen Prüfung: 24.11.2011 2 3 Meiner Mutter in Dankbarkeit 4 Index 1 INDEX 1  INDEX ..................................................................................................................................... 5 2  ABBREVIATIONS ................................................................................................................... 8 3  INTRODUCTION .................................................................................................................... 9 3.1  BACKGROUND ..............................................................................................................

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 33
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

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Aus der Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen Kinderspital der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. med. Ch. Klein

Abteilung Hämatologie/Onkologie 

Downregulation of endogenous TRAIL and its effect on the human cancer cell line KELLY  Dissertation zum Erwerb des Doktorgrades der Medizin an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München  

 vorgelegt von Sara Brittingham aus Warschau 2011

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Mit Genehmigung der Medizinischen Fakultät der Universität München    

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PD Dr. Andreas Jung Prof. Dr. Friedrich-Wilhelm Kreth

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 Berichterstatter:  Mitberichterstatter:      Dekan:  Tag der mündlichen Prüfung:   

24.11.2011

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PD Dr. Irmela Jeremias

Prof.Dr.Dr.h.c.M.Reiser;FACR,FRCR

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Meiner Mutter in Dankbarkeit

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Index

1 INDEX 

1 .......................................................................................IEXND............................................5..  2   ................8....................................................................AREBBATVINSIO...............................   3    9INTRODUCTION ....................................................................................................................   3.1   B ACKGROUND ...................................................................................................................... 9   3.2   M ULTIPLE BIOLOGICAL FUNCTIONS OF TRAIL........................................................................ 9   3.3   E XPRESSION OF T UMOR NECROSIS FACTOR - RELATED APOPTOSIS - INDUCING LIGAND (TRAIL).....................................................................................................................................10   3.4   TRAIL AND ITS RECEPTORS ............................................................................................... 11   3.5   A POPTOSIS AND THE PRO - APOPTOTIC EFFECT OF  11TRAIL ....................................................   3.6   TRAIL AS AN ANTI - CANCER DRUG ..............................................................................13.........   3.7   N ON - APOPTOTIC EFFECT OF TRAIL.................................................................................... 14   3.8   T HE PRO - PROLIFERATIVE EFFECT OF TRAIL ....................................................................... 15   3.9   A IM OF PRESENT WORK ....................................................................................................1..6   4   ....................................................................................17.................................ETAMLAIR.......   4.1   C ELL LINES ........................................................71................................................................   4.2   O LIGONUCLEOTIDES .......................................................................................................... 18   4.3   A NTIBODIES OF W ESTERN BLOT ANALYSIS ................91..........................................................   4.4   K ITS ........................................19..........................................................................................   4.5   S OFTWARE .......91.................................................................................................................   4.6   L ABORATORY E QUIPEMENT 19................................................................................................   4.7   M ATERIALS OF CELL CULTURE .....20.......................................................................................   4.8   C HEMICALS ....................................................................................................................... 21   4.9   S OLUTIONS / BUFFERS .......................................................................22.................................   5   ...32M........................................ETHODS.................................................................................   5.1   C ELL CULTURE ...........................................................................................................23........   5.2   I N - VITRO ASSAYS ............................................................................................................... 23   5.2.1   Stimulation with TRAIL..................................................................................23   5.2.2   Transient transfection....................................................................................24   5.2.3   Caspase inhibition.........................................................................................25   5

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Index

5.3 C ELL GROWTH ANALYSIS USING C ELL S CREEN .................................................................... 25  5.4   C ELL DEATH ANALYSIS USING F LUORESCENCE ACTIVATED CELL SORTING  26(FACS) ...............   5.5   A NALYSIS OF TRANSFECTION EFFICIENCY USING F LUORESCENCE ACTIVATED CELL SORTING (FACS) ....................................................................................................................... 27   5.6   S TATISTICAL A NALYSIS ...................................................................................................... 27   5.7   RNA/DNA STUDIES ........................................................................................................... 27   5.7.1   RNA Isolation and quantification ...................................................................27   5.7.2   Reverse Transcriptase Reaction...................................................................28   5.7.3    .................29Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)   5.7.4   Analysis of qRT-PCR (crossing point and melting curve analysis) ...............30   5.8   P ROTEIN STUDIES ..........................................................................................23....................   5.8.1    ........................................................................................................32Cell lysis   5.8.2   SDS-Polyacrylamid Gel Electrophoresis (PAGE) .........................................33   5.8.3    ..........................................................34Immunoblotting and Immunodetection   6   RESULTS ............................................................................................................................. 35   6.1   TRAIL MEDIATED PROLIFERATION ...................................................................................... 35   6.2   D OWNREGULATION OF ENDOGENOUS TRAIL: OPTIMIZATION ................................................ 37   6.2.1   Screening human cell lines for endogenous TRAIL expression....................37   6.2.2   Evaluation of siRNA delivery.........................................................................39   6.2.3   Evaluation of siRNA efficiency ......................................................................41   6.2.3.1   Efficiency on RNA level .............................................................................41   6.2.3.2   Efficiency on Protein Level ........................................................................43   6.3   D OWNREGULATION OF ENDOGENOUS TRAIL: SCREENING FOR EFFECTS ON CELL GROWTH ............................44........................................................................................................   6.4   KELLY CELLS AND THE EFFECTS OF DOWNREGULATION OF ENDOGENOUS TRAIL................ 50   6.4.1   Reduction of cell growth................................................................................50   6.4.2   Cell growth reduction is a result of cell death induction ................................53   6.4.3   Soluble TRAIL rescues cell death induced by the downregulation of endogenous TRAIL......................................................................................................56   6.4.4   Cell death is mediated by caspases..............................................................58   7    61DISCUSSION .......................................................................................................................   7.1   S UBSTANTIAL EFFECT OF THE DOWN REGULATION OF TRAIL ON CELL VIABILITY ................61...   6

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Index

7.2 C ELL - TYPE SPECIFICITY OF THE FUNCTION OF ENDOGENOUS TRAIL .................................... 62  7.3   R OLE OF ENDOGENOUS TRAIL FOR CANCER CELLS ............................................................ 63   7.4   P RO - SURVIVAL FUNCTION OF SEVERAL MEMBERS OF THE TNF- FAMILY ................................ 64   7.5   M ECHANISMS OF THE PRO - SURVIVAL FUNCTION OF TNF- FAMILY MEMBERS ..........................65   7.6   F UTURE PERSPECTIVE ....................................................................................................... 65   7.7   P OTENTIAL RELEVANCE AS A NOVEL APPROACH OF CANCER THERAPY ................................. 66  

8 SUMMARY ........................................................................................................................... 67 

9 ...86................................................................FNEMUSSA.GN....ZAMUS................................ 

10 REFERENCES ..................................................................................................................... 69 

11 ACKNOWLEDGEMENT ....................................................................................................... 78 

12 ................................................................PON.....UBLICATI................................97................. 

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